Formulations of VEG antagonist fusion proteins and method of manufacturing them

ABSTRACT

Formulations of a vascular endothelial growth factor (VEGF)-specific fusion protein antagonist are provided including a pre-lyophilized formulation, a reconstituted lyophilized formulation, and a stable liquid formulation. Preferably, the fusion protein has the sequence of SEQ ID NO:4.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/342,989, filed on Nov. 3, 2016, which is a continuation of U.S.patent application Ser. No. 15/064,343, filed on Mar. 8, 2016 andgranted on Dec. 6, 2016 as U.S. Pat. No. 9,511,140, which is acontinuation of U.S. patent application Ser. No. 14/550,385, filed onNov. 21, 2014 and granted on Aug. 16, 2016 as U.S. Pat. No. 9,416,167,which is a continuation of U.S. patent application Ser. No. 13/909,745,filed on Jun. 4, 2013 and granted on Dec. 30, 2014 as U.S. Pat. No.8,921,316, which is a continuation of U.S. patent application Ser. No.13/428,510, filed on Mar. 23, 2012 and granted on Apr. 29, 2014 as U.S.Pat. No. 8,710,004, which is a continuation of U.S. patent applicationSer. No. 13/343,214, filed on Jan. 4, 2012 and granted on Mar. 26, 2013as U.S. Pat. No. 8,404,638, which is a division of U.S. patentapplication Ser. No. 12/835,065, filed on Jul. 13, 2010 and granted onFeb. 7, 2012 as U.S. Pat. No. 8,110,546, which is a continuation of U.S.patent application Ser. No. 11/387,256, filed on Mar. 22, 2006, whichclaims the benefit of priority under 35 USC § 119(e) of U.S. ProvisionalApplication No. 60/665,125, filed on Mar. 25, 2005, all of which areherein specifically incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is directed to pharmaceutical formulationscomprising agents capable of inhibiting vascular endothelial growthfactor (VEGF), and to methods for making and using such formulations.The invention includes pharmaceutical formulations having increasedstability.

Statement of Related Art

Vascular endothelial growth factor (VEGF) expression is nearlyubiquitous in human cancer, consistent with its role as a key mediatorof tumor neoangiogenesis. Blockade of VEGF function, by binding to themolecule or its VEGFR-2 receptor, inhibits growth of implanted tumorcells in multiple different xenograft models (see, for example, Gerberet al. (2000) Cancer Res. 60:6253-6258). A soluble VEGF-specific fusionprotein antagonist, termed a “VEGF trap” has been described (Kim et al.(2002) Proc. Natl. Acad. Sci. USA 99:11399-404; Holash et al. (2002)Proc. Natl. Acad. Sci. USA 99:11393-8), which references arespecifically incorporated by reference in their entirety.

Lyophilization (freeze drying under controlled conditions) is commonlyused for long term storage of proteins. The lyophilized protein issubstsntially resistant to degradation, aggregation, oxidation, andother degenerative processes while in the freeze-dried state (see, forexample, U.S. Pat. No. 6,436,897).

BRIEF SUMMARY OF THE INVENTION

Stable formulations of a VEGF-specific fusion protein antagonist areherein provided. The pharmaceutically acceptable formulations of theinvention comprise the VEGF “trap” antagonist with a pharmaceuticallyacceptable carrier. In specific embodiments, liquid and freeze-dried, orlyophilized formulations are provided.

In a first aspect, the invention features a stable liquid formulation ofa VEGF-specific fusion protein antagonist, comprising a fusion proteincomprising a receptor component consisting essentially of animmunoglobulin-like (Ig) domain 2 of a first VEGF receptor and Ig domain3 of a second VEGF receptor, and a multimerizing component, one or morebuffers, and one or more thermal stabilizers. In a specific embodimentof the VEGF-specific fusion protein antagonist, the first VEGF receptoris Flt1 and the second VEGF receptor is Flk1 or Flt4. In a more specificembodiment the fusion protein has the amino acid sequence of SEQ ID NO:2or SEQ ID NO:4. In one embodiment, the buffer is a phosphate bufferand/or citrate. More preferably, the buffers are phosphate and citrate.In one embodiment, the thermal stabilizers are NaCl and/or sucrose. Morepreferably, the thermal stabilizers are both NaCl and sucrose.

In a specific embodiment, the stable liquid formulation of aVEGF-specific fusion protein antagonist comprises 1-10 mM phosphatebuffer, 1-10 mM citrate, 25-150 mM NaCl, 5-30% sucrose, 10-50 mg/ml ofthe fusion protein, at a pH of about 6-6.5. In a more specificembodiment, the stable liquid formulation comprises 5 mM phosphatebuffer, 5 mM citrate buffer, 100 mM NaCl, 20% sucrose, 25 mg/ml of thefusion protein, at a pH of about 6.0. Additionally, polysorbate may bepresent, for example 0.05-0.15% polysorbate 20. The stable liquidformulation of the VEGF-specific fusion protein antagonist of theinvention exhibits little or no precipitation after storage of a 25mg/ml VEGF formulation for about 6 months at −80° C. and little or noprecipitation after storage for 6 months at 5° C.

In a second aspect, the invention features a high concentration stableliquid formulation of a VEGF antagonist comprising 1-50 mM histidine,25-150 mM NaCl, 5-30% sucrose, 50-100 mg/ml of the fusion protein, at apH of about 6-6.5, and either 0.1-0.5% polysorbate or 1-5% PEG. In amore specific embodiment, the high concentration stable liquidformulation comprises 10 mM histidine, 50 mM NaCl, 5-20% sucrose, 50-100mg/ml of the fusion protein, at a pH of about 6.0-6.5, with either 0.1%polysorbate (e.g., polysorbate 20) or 3% PEG (e.g., PEG 3350). The highconcentration stable liquid formulation of the VEGF-specific fusionprotein antagonist of the invention exhibits less than about 3%degradation after 15 months of storage at 5° C. (75 or 100 mg/ml VEGFtrap protein) or less than about 1.5% degradation after 24 months (50mg/ml).

In a third aspect, the invention features a pre-lyophilized formulationof a vascular endothelial growth factor (VEGF)-specific fusion proteinantagonist, comprising a (i) fusion protein comprising a receptorcomponent consisting essentially of an immunoglobulin-like (Ig) domain 2of a first VEGF receptor and Ig domain 3 of a second VEGF receptor, anda multimerizing component, (ii) a buffer, (iii) an organic co-solvent orbulking agent, and (iv) one or more lyoprotectants. In variousembodiments, the buffer is histidine, the organic co-solvent or bulkingagent is PEG, and the lyoprotectant(s) is at least one of glycine andsucrose. In one embodiment, the pre-lyophilized formulation of theinvention does not contain a preservative.

In one embodiment of the pre-lyophilized formulation of the invention,the formulation comprises 5-50 mM histidine, 0.1-3.0% PEG, 0.25-3.0%glycine, 0.5-6.0% sucrose, and 5-75 mg/ml of the fusion protein, at a pHof about 6.0-6.5. In any embodiment, the pre-lyophilized formulation mayfurther comprise up to 0.05 mM citrate and/or 0.003-0.005% polysorbate.The polysorbate present may be, for example, polysorbate 20.

In a more specific embodiment, the pre-lyophilized formulation comprisesabout 10 mM histidine, about 1.5% PEG 3350, about 0.75% glycine, about2.5% sucrose, and about 12.5 to 75 mg/ml VEGF-specific fusion protein,at a pH of about 6.25. In specific embodiments, the fusion proteincomprises the protein sequence of SEQ ID NO:4, present as a multimer,e.g., a dimer. In separate embodiments, the reconstituted formulation is2 times the concentration of the pre-lyophilized formulation, e.g., a 20mg fusion protein/ml pre-lyophilized formulation is reconstituted to afinal formulation of 60 mg fusion protein/mi. Generally, the lyophilizedformulation is reconstituted with sterile water suitable for injection.In one embodiment, the reconstitution liquid may be bacteriostaticwater.

In a preferred embodiment, the pre-lyophilized formulation consistsessentially of about 10 mM histidine, about 1.5% PEG 3350, about 0.75%glycine, about 2.5% sucrose, and about 50 mg/ml of the fusion proteinhaving the sequence of SEQ ID NO:4 as a dimer, at a pH of about 6.25.Citrate (less than or equal to about 0.02 mM) and/or polysorbate (lessthan or equal to about 0.0005%) may be present. Optionally, thepre-lyophilized formulation does not contain a preservative, a phosphatebuffer, and/or more than trace amounts of NaCl. In one embodiment, thepre-lyophilized formulation consists of about 10 mM histidine, about1.5% PEG 3350, about 0.75% glycine, about 2.5% sucrose, and about 50mg/ml of the VEGF trap protein (SEQ ID NO:4), pH 6.3, and uponreconstitution contains 20 mM histidine, 3% PEG, 1.5% glycine, about 5%sucrose, and about 100 mg/ml VEGF trap protein.

In a fourth aspect, the invention features a method of producing alyophilized formulation of a VEGF-specific fusion protein antagonist,comprising subjecting the pre-lyophilized formulation of the inventionto lyophilization to generate a lyophilized formulation. The lyophilizedformulation may be lyophilized by any method known in the art forlyophilizing a liquid.

In a fifth related aspect, the invention features a method of producinga reconstituted lyophilized formulation of a VEGF-specific fusionprotein antagonist, comprising reconstituting the lyophilizedformulation of the invention to a reconstituted formulation. In oneembodiment, the reconstituted formulation is twice the concentration ofthe pre-lyophilized formulation, e.g., the method of the inventioncomprises: (a) producing a pre-lyophilized formulation of aVEGF-specific fusion protein antagonist, (b) subjecting thepre-lyophilized formulation of step (a) to lyophilization; and (c)reconstituting the lyophilized formulation of step (b).

In specific embodiments of the method of producing a reconstitutedlyophilized formulation, a pre-lyophilized solution is present in a vialas a 25 mg VEGF-specific fusion protein antagonist per ml solution ofpre-lyophilized formulation, which is lyophilized and reconstituted toan 50 mg/ml solution. In another embodiment, a 30 mg/ml pre-lyophilizedsolution is lyophilized and reconstituted to a 60 mg/ml solution. Inanother embodiment, a 40 mg/ml pre-lyophilized solution is lyophilizedand reconstituted to a 80 mg/ml solution. In another embodiment, a 12.5mg/ml pre-lyophilized solution is lyophilized and reconstituted to a 25mg/ml solution. In another embodiment, a 50 mg/ml pre-lyophilizedsolution is lyophilized and reconstituted to a 100 mg/ml solution. Inanother embodiment, a 75 mg/ml pre-lyophilized solution is lyophilizedand reconstituted to a 150 mg/ml solution. Preferably, the reconstitutedlyophilized formulation does not contain a preservative.

Other objects and advantages will become apparent from a review of theensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is not limited to particular methods, andexperimental conditions described, as such methods and conditions mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting unless indicated, since the scope of the presentinvention will be limited only by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “a method”include one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

Unless stated otherwise, all technical and scientific terms and phrasesused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention belongs. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference.

General Description

Safe handling and administration of formulations comprising proteinsrepresent significant challenges to pharmaceutical formulators. Proteinspossess unique chemical and physical properties that present stabilityproblems: a variety of degradation pathways exist for proteins,implicating both chemical and physical instability. Chemical instabilityincludes deamination, aggregation, clipping of the peptide backbone, andoxidation of methionine residues. Physical instability encompasses manyphenomena, including, for example, aggregation.

Chemical and physical stability can be promoted by removing water fromthe protein. Lyophilization (freeze-drying under controlled conditions)is commonly used for long-term storage of proteins. The lyophilizedprotein is substantially resistant to degradation, aggregation,oxidation, and other degenerative processes while in the freeze-driedstate. The lyophilized protein is normally reconstituted with wateroptionally containing a bacteriostatic preservative (e.g., benzylalcohol) prior to administration.

Definitions

The term “carrier” includes a diluent, adjuvant, excipient, or vehiclewith which a composition is administered. Carriers can include sterileliquids, such as, for example, water and oils, including oils ofpetroleum, animal, vegetable or synthetic origin, such as, for example,peanut oil, soybean oil, mineral oil, sesame oil and the like.

The term “excipient” includes a non-therapeutic agent added to apharmaceutical composition to provide a desired consistency orstabilizing effect. Suitable pharmaceutical excipients include, forexample, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like.

The term “lyophilized” or “freeze-dried” includes a state of a substancethat has been subjected to a drying procedure such as lyophilization,where at least 50% of moisture has been removed.

The phrase “bulking agent” includes a compound that is pharmaceuticallyacceptable and that adds bulk to a lyo cake. Generally, acceptablebulking agents known to the art include, for example, carbohydrates,including simple sugars such as dextrose, ribose, fructose and the like,alcohol sugars such as mannitol, inositol and sorbitol, disaccharidesincluding trehalose, sucrose and lactose, naturally occurring polymerssuch as starch, dextrans, chitosan, hyaluronate, proteins (e.g, gelatinand serum albumin), glycogen, and synthetic monomers and polymers. Inthe formulations of the invention, PEG 3350 is an organic co-solventwhich is used to stabilize the fusion protein when agitated, mixed, orhandled, and as a bulking agent to help produce an acceptable bulk.

The term “lyoprotectant” includes a substance that may be added to afreeze-dried or lyophilized formulation to help maintain proteinstructure when freeze-dried or lyophilized.

A “preservative” includes a bacteriostatic, bacteriocidal, fungistaticor fungicidal compound that is generally added to formulations to retardor eliminate growth of bacteria or other contaminating microorganisms inthe formulations. Preservatives include, for example, benzyl alcohol,phenol, benzalkonium chloride, m-cresol, thimerosol, chlorobutanol,methylparaben, propylparaben and the like. Other examples ofpharmaceutically acceptable preservatives can be found in the USP.

VEGF Antagonists

An VEGF antagonist is a compound capable of blocking or inhibiting thebiological action of vascular endothelial growth factor (VEGF), andincludes fusion proteins capable of trapping VEGF. In a preferredembodiment, the VEGF antagonist is the fusion protein of SEQ ID NO:2 or4; more preferably, SEQ ID NO:4. In specific embodiments, the VEGFantagonist is expressed in a mammalian cell line such as a CHO cell andmay be modified posttranslationally. In a specific embodiment, thefusion protein comprises amino acids 27-457 of SEQ ID NO:4 and isglycosylated at Asn residues 62, 94, 149, 222 and 308.

The VEGF antagonist of the methods and formulations of the invention canbe prepared by any suitable method known in the art, or that comes to beknown. The VEGF antagonist is preferably substantially free of proteincontaminants at the time it is used to prepare the pharmaceuticallyacceptable formulation. By “substantially free of protein contaminants”is meant, preferably, that at least 90% of the weight of protein of theVEGF-specific fusion protein antagonist preparation used for making aformulation is VEGF fusion protein antagonist protein, more preferablyat least 95%, most preferably at least 99%. The fusion protein ispreferably substantially free of aggregates. “Substantially free ofaggregates” means that at least 90% of the weight of fusion protein isnot present in an aggregate at the time the fusion protein is used toprepare the pharmaceutically effective formulation. The fusion proteinof the methods and formulations of the invention may contain low ortrace amounts of compounds as a results of the purification process, forexample, low or trace amounts of citrate and/or polysorbate. In oneembodiment of the pre-lyophilized formulation of the inventioncontaining about 50 mg of fusion protein/ml, citrate may be present at aconcentration of about 0.02 mM and/or polysorbate may be present at aconcentration of about 0.0005%. If the pre-lyophilized formulation isreconstituted after lyophilization to half of the original volume (e.g.,100 mg/ml of fusion protein), the resulting concentrations may be 0.04mM citrate and/or 0.001% polysorbate.

Lyophilization and Lyophilized Formulations

In one aspect of the invention, a pharmaceutically acceptableformulation comprising a VEGF-specific fusion protein antagonist isprovided, wherein the formulation is a freeze-dried or lyophilizedformulation. Lyophilized formulations can be reconstituted intosolutions, suspensions, emulsions, or any other suitable form foradministration or use. Lyophilized formulations are typically firstprepared as liquids, then frozen and lyophilized. The total liquidvolume before lyophilization can be less, equal to, or more than, thefinal reconstituted volume of the lyophilized formulation. Thelyophilization process is well known to those of ordinary skill in theart, and typically includes sublimation of water from a frozenformulation under controlled conditions.

Lyophilized formulations can be stored at a wide range of temperatures.Lyophilized formulations may be stored below 25° C., for example,refrigerated at 4° C., or at room temperature (e.g., approximately 25°C.). Preferably, lyophilized formulations are stored below about 25° C.,more preferably, at about 4-20° C.; below about 4° C.; below about −20°C.; about −40° C.; about −70° C., or about −80° C.

Lyophilized formulations are typically reconstituted for use by additionof an aqueous solution to dissolve the lyophilized formulation. A widevariety of aqueous solutions can be used to reconstitute a lyophilizedformulation. Preferably, lyophilized formulations are reconstitutedusing water. Lyophilized formulations are preferably reconstituted witha solution consisting essentially of water (e.g., USP WFI, or water forinjection) or bacteriostatic water (e.g., USP WFI with 0.9% benzylalcohol). However, solutions comprising buffers and/or excipients and/orone or more pharmaceutically acceptable carries can also be used.

Freeze-dried or lyophilized formulations are typically prepared fromliquids, that is, from solutions, suspensions, emulsions, and the like.Thus, the liquid that is to undergo freeze-drying or lyophilizationpreferably comprises all components desired in a final reconstitutedliquid formulation. As a result, when reconstituted, the freeze-dried orlyophilized formulation will render a desired liquid formulation uponreconstitution. A preferred liquid formulation used to generate afreeze-dried or lyophilized formulation comprises a VEGF-specific fusionprotein antagonist in a pharmaceutically effective amount, a buffer, astabilizer, and a bulking agent. Freeze-dried or lyophilizedformulations preferably comprise histidine, since histidine, incomparison to phosphate, is more effective at stabilizing the fusionprotein when the fusion protein is lyophilized. Organic co-solvents,such as PEG 3350, are used to stabilize the fusion protein whenagitated, mixed, or handled. A lyoprotectant is preferably used infreeze-dried or lyophilized formulations. Lyoprotectants help tomaintain the secondary structure of proteins when freeze-dried orlyophilized. Two preferred example lyoprotectants are glycine andsucrose, which are preferably used together.

Stable Liquid Formulations

In one aspect, the invention provides a stable pharmaceuticallyacceptable formulation comprising a VEGF-specific fusion proteinantagonist, wherein the formulation is a liquid formulation. Preferably,the liquid formulation comprises a pharmaceutically effective amount ofthe fusion protein. The formulation can also comprise one or morepharmaceutically acceptable carriers, buffers, bulking agents,stabilizers, preservatives, and/or excipients. An example of apharmaceutically acceptable liquid formulation comprises a VEGF-specificfusion protein antagonist in a pharmaceutically effective amount, abuffer, a co-solvent, and one or more stabilizers.

A preferred liquid formulation comprises phosphate buffer, an organicco-solvent, and one or more thermal stabilizers to minimize formation ofaggregates and low molecular weight products when stored, and about 10mg/ml to about 50 mg/ml fusion protein, wherein the formulation is fromabout pH 6.0-6.5. A preferred liquid formulation comprises about 5 mMphosphate buffer, about 5 mM citrate, about 100 mM NaCl, about 25%sucrose, and about 1050 mg/ml fusion protein, wherein the formulation isat a pH of about 6.0; optionally polysorbate may be present (e.g., 0.1%polysorbate 20). Although either NaCl or sucrose can be used as astabilizer, a combination of NaCl and sucrose has been established tostabilize the fusion protein more effectively than either individualstabilizer alone.

Stability is determined in a number of ways at specified time points,including determination of pH, visual inspection of color andappearance, determination of total protein content by methods known inthe art, e.g., UV spectroscopy, SDS-PAGE, size-exclusion HPLC, bioassaydetermination of activity, isoelectric focusing, and isoaspartatequantification. In one example of a bioassay useful for determining VEGFantagonist activity, a BAF/3 VEGFR1/EPOR cell line is used to determineVEGF165 binding by the VEGF-specific fusion protein antagonist of theinvention.

Formulations, whether liquid or freeze-dried and lyophilized, can bestored in an oxygen-deprived environment. Oxygen-deprived environmentscan be generated by storing the formulations under an inert gas such as,for example, argon, nitrogen, or helium.

EXAMPLES

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly to the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described.

Example 1. Stability of a 50 mg/ml Liquid Formulation of VEGF Trap

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 0.1%polysorbate 20, 20% sucrose, and 50 mg/ml VEGF trap (SEQ ID NO:4), pH6.25, was stored at 5° C. and samples tested at 3, 6, 9, 12, 18 and 24months. Stability was determined by SE-HPLC. The results, shown in Table1, show that 98.6% and 98.3% of VEGF trap protein remained intact(non-degraded) at 12 and 24 months, respectively. Turbidity was measuredat OD405 nm; and percent recovered protein by size exclusion HPLC.

TABLE 1 Stability of 50 mg/ml VEGF Trap Protein When Stored at 5° C.(VGFT-SS065) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.2 100 99.0 3 Pass0.00 6.2 102 98.8 6 Pass 0.01 6.2 103 98.7 9 Pass 0.01 6.3 102 98.2 12Pass 0.01 6.3 106 98.6 18 Pass 0.00 6.3 103 98.4 24 Pass 0.00 6.2 9398.3

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 3% PEG3350, 20% sucrose, and 50 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, wasstored at 5° C. and samples tested at 3, 6, 9, 12, 18 and 24 months.Stability results are shown in Table 2.

TABLE 2 Stability of 50 mg/ml VEGF Trap Protein When Stored at 5° C.(VGFT-SS065) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.2 100 99.0 3 Pass0.00 6.2 100 98.8 6 Pass 0.01 6.3 103 98.5 9 Pass 0.00 6.3 103 98.3 12Pass 0.01 6.3 110 98.3 18 Pass 0.00 6.3 113 98.0 24 Pass 0.01 6.2 9097.8

Example 2. Stability of a 75 mg/ml Liquid Formulation of VEGF Trap

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 0.1%polysorbate 20, 20% sucrose, and 75 mg/ml VEGF trap (SEQ ID NO:4), pH6.25, was stored at 5° C. and samples tested at 0, 1, 2.3, 3, 9, 12 and15 months. Stability results are shown in Table 3.

TABLE 3 Stability of 75 mg/ml VEGF Trap Protein When Stored at 5° C.(VGFT-SS101) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.2 100 97.1 1 Pass0.00 6.2 96 97.0 2.3 Pass 0.00 6.2 98 96.7 3 Pass 0.00 6.2 97 96.1 9Pass −0.01 6.0 101 96.0 12 Pass 0.00 6.3 110 94.5 15 Pass 0.00 6.3 9295.6

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 3% PEG3350, 20% sucrose, and 75 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, wasstored at 5° C. and samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months.Stability results are shown in Table 4.

TABLE 4 Stability of 75 mg/ml VEGF Trap Protein When Stored at 5° C.(VGFT-SS101) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.2 100 96.8 1 Pass0.00 6.2 99 96.7 2.3 Pass 0.00 6.2 97 96.3 3 Pass 0.00 6.2 89 95.6 9Pass −0.01 6.2 98 95.4 12 Pass −0.01 6.3 112 94.1 15 Pass 0.00 6.3 9894.8

Example 3. Stability of a 100 mg/ml Liquid Formulation of VEGF Trap

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 0.1%polysorbate 20, 20% sucrose, and 100 mg/ml VEGF trap (SEQ ID NO:4), pH6.25, was stored at 5° C. and samples tested at 0, 1, 2.3, 3, 9, 12 and15 months. Stability results are shown in Table 5.

TABLE 5 Stability of 100 mg/ml VEGF Trap Protein Stored at 5° C.(VGFT-SS101) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.3 100 96.7 1 Pass0.00 6.2 92 96.6 2.3 Pass 0.00 6.2 92 96.2 6 Pass 0.00 6.2 99 95.5 9Pass −0.01 6.2 92 95.5 12 Pass −0.01 6.2 110 93.9 15 Pass 0.00 6.3 10894.8

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 3% PEG3350, 20% sucrose, and 100 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, wasstored at 5° C. and samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months.Stability results are shown in Table 6.

TABLE 6 Stability of 100 mg/ml VEGF Trap Protein Stored at 5° C.(VGFT-SS101) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.3 100 96.5 1 Pass0.01 6.2 94 96.2 2.3 Pass 0.01 6.2 93 95.7 6 Pass 0.01 6.2 102 94.6 9Pass 0.00 6.2 95 94.6 12 Pass 0.00 6.3 96 92.8 15 Pass 0.01 6.3 102 93.9

Example 4. Further Embodiments of Stable VEGF Trap Formulations

In one embodiment, the invention provides a stable liquid VEGF-bindingfusion protein (VEGF trap) formulations comprising 5 mM phosphate, 5 mMcitrate, 100 mM NaCl, 0.1% Polysorbate 20, 20% sucrose, 25 mg/ml VEGFtrap protein, pH 6.0. This formulation can either be deliveredsubcutaneously or diluted and delivered by intravenous infusion. Due tothe high osmolality of this formulation, it is diluted 3-fold to achievean iso-osmolar solution for intravenous administration. Stabilitystudies showed less than about 1% degradation was detected after 3 yearsof storage at 2-8° C.

In one embodiment, the invention features a lyophilized formulationwhich is preferably concentrated two-fold from the pre-lyophilized tothe post-lyophilized formulation, e.g., 50 to 100 mg/ml; 75 to 150mg/ml, or 100 to 200 mg/ml VEGF trap protein. In one specificembodiment, the pre-lyophilized formulation comprises 10 mM histidine,1.5% PEG 3350, 0.75% glycine, 2.5% sucrose, 50 mg/ml VEGF trap protein,pH 6.3, and is reconstituted to a formulation comprising 20 mMhistidine, 3% PEG 3350, 1.5% glycine, 5% sucrose, 100 mg/ml VEGF trapprotein, pH 6.3. Stability studied showed no degradation of the VEGFtrap was detected after 6 months of storage at 2-8° C.

In one embodiment of a liquid formulation, the formulation comprises 10mM histidine, 50 mM NaCl, 5-20% sucrose, 50-100 mg/ml VEGF trap, and oneof 0.1% polysorbate 20 or TY° PEG 3350. One advantage of this liquidformulation is that it provides a higher concentration of VEGF trapwithout requiring the manufacture of a lyophilized product. Thus, thisformulation provides ease for subcutaneous delivery, for example, byallowing provision of a liquid pre-filled syringe at a concentrationhigher than that delivered by IV infusion. Also, this formulation couldadvantageously be used to provide lower infusion volumes and shorterinfusion times. The amount of degradation determined by SE-HPLCfollowing incubation at 5° C. for up to 15 or 24 months is summarized inTable 7.

TABLE 7 Stability of Liquid Formulation with 50-100 mg/ml VEGF Trap(VGFT-SS101) Incubation VEGF Trap % Polysorbate % PEG (months) (mg/ml)20 3350 % Degradation 24 50 0.1 — 0.7 24 50 — 3 1.3 15 75 0.1 — 1.5 1575 — 3 2.0 15 100 0.1 — 1.9 15 100 — 3 2.6

Example 5. Stability and Activity of Lyophilized and Liquid

The stability of a reconstituted lyophilized formulation was determinedover a 6 month period. The pre-lyophilized formulation contained 10 mMhistidine, 1.5% PEG 3350, 2.5% sucrose, 0.75% glycine and 50 mg/ml VEGFtrap protein. After lyophilization, the reconstituted formulationcontained 20 mM histidine, 3% PEG 3350, 5% sucrose, 1.5% glycine, and100 mg/ml VEGF trap protein (SEQ ID NO:4). The results are shown inTable 8. Activity was determined in a cell based bioassay which directlymeasures the ability of the VEGF trap to inhibit the biological effectsof human VEGF on a mouse Baf/3 VEGFR1/EpoR cell line. Therefore, thisbioassay directly measures the biological activity of the protein. Theresults are expresses as percent relative potency (test sampleIC₅0/reference VEGF IC₅0 standard×100). The binding affinity of VEGF tothe VEGF trap is measured using a sensitive ELISA that specificallymeasures free VEGF in equilibrated mixtures containing VEGF and variousconcentrations of the VEGF trap. Results are expressed as percentrelative binding (IC₅0 of test sample/ICH, of reference×100). MeasuredpH ranged between 6.3-6.5. All solutions where visually clear. Theconcentration of VEGF trap recovered was determined with a UVspectrophotometer as mg/ml at A280 nm. The percent VEGF trap recoveredin the native configuration (main peak purity) was determined withSE-HPLC.

TABLE 8 Stability of VEGF Trap Lyophilized Formulation Stored at 5° C.(VGT-RS475) Binding % Native Months Bioassay Assay % RecoveredConfiguration 0 120 126 97.9 98.7 1 117 74 97.9 98.6 1 + 24 hr 126 7299.0 98.5  1 + 4 hr 94 81 101.5 98.2 3 101 98 98.1 98.6 3 + 24 hr 65 9498.1 98.2  6 + 4 hr 96.9 98.7 6 + 24 hr 98.8 98.6

A formulation containing about 5 mM phosphate, 5 mM citrate, 100 mMNaCl, 0.1% polysorbate 20, 20% sucrose, and 25 mg/ml VEGF trap proteinwas tested for stability and activity over 36 months when stored at 5°C. The results are shown in Table 9. All samples were clear andcolorless as determined by visual inspection. pH ranged from 6.0-6.1.*Binding assay results for two measurements (1 and 2 months) areexpressed directly and not as a percent of the standard.

TABLE 9 Stability and Activity of Liquid Formulation (VGT-FS405) %Native Binding Protein Content Months Configuration Bioassay Assay mg/ml0 99.7 106 72 25.0 1 99.9 119 4.4 ρM* 25.2 2 99.6 102 5.4 ρM* 25.1 399.6 97 88 25.1 6 99.6 101 106  25.0 9 99.4 89 126  25.4 12 99.5 85 9525.2 18 99.4 99 81 25.5 24 99.3 75 95 25.6 36 98.8 109 79 25.6

We claim:
 1. A formulation comprising: 10-50 mg/ml of a vascularendothelial growth factor (VEGF) antagonist fusion protein comprisingamino acids 27-457 of SEQ ID NO: 4, a buffer comprising histidine, anorganic co-solvent comprising polysorbate, and a stabilizing agentcomprising a sugar, an amino acid or both, wherein said VEGF antagonistfusion protein exhibits less than about 3% degradation after 15 monthsof storage at 5° C.
 2. The formulation of claim 1, wherein said buffercomprises 5-50 mM histidine.
 3. The formulation of claim 1, wherein saidbuffer comprises about 10 mM histidine.
 4. The formulation of claim 1,wherein said buffer is at a pH of about 6.0-6.5.
 5. The formulation ofclaim 2, wherein said formulation comprises 40 mg/ml of said VEGFantagonist fusion protein.
 6. The formulation of claim 2, wherein saidformulation further comprises a tonicity agent.
 7. The formulation ofclaim 6, wherein said tonicity agent is sodium chloride.
 8. Theformulation of claim 2, wherein said organic co-solvent comprises0.1-0.5% polysorbate.
 9. The formulation of claim 2, wherein saidorganic co-solvent comprises polysorbate
 20. 10. The formulation ofclaim 2, wherein said stabilizing agent comprises a sugar.
 11. Theformulation of claim 10, wherein said sugar is selected from groupconsisting of dextrose, ribose, fructose, mannitol, inositol, sorbitol,trehalose, sucrose, and lactose.
 12. The formulation of claim 10,wherein said sugar is 5-20% sucrose.
 13. The formulation of claim 10,wherein said sugar is 0.5-6.0% sucrose.
 14. The formulation of claim 2,wherein said stabilizing agent comprises an amino acid.
 15. Theformulation of claim 14, wherein said amino acid is glycine.
 16. Theformulation of claim 14, wherein said amino acid is 0.25-3.0% glycine.17. The formulation of claim 9, wherein said formulation comprises 40mg/ml of said VEGF antagonist fusion protein.
 18. The formulation ofclaim 9, wherein said formulation further comprises a tonicity agent.19. The formulation of claim 18, wherein said tonicity agent is sodiumchloride.
 20. The formulation of claim 9, wherein said stabilizing agentcomprises a sugar.
 21. The formulation of claim 20, wherein said sugaris selected from group consisting of dextrose, ribose, fructose,mannitol, inositol, sorbitol, trehalose, sucrose, and lactose.
 22. Theformulation of claim 20, wherein said sugar is 5-20% sucrose.
 23. Theformulation of claim 20, wherein said sugar is 0.5-6.0% sucrose.
 24. Theformulation of claim 9, wherein said stabilizing agent comprises anamino acid.
 25. The formulation of claim 24, wherein said amino acid isglycine.
 26. The formulation of claim 24, wherein said amino acid is0.25-3.0% glycine.
 27. A formulation comprising: 10-50 mg/ml of avascular endothelial growth factor (VEGF) antagonist fusion proteincomprising amino acids 27-457 of SEQ ID NO: 4, a buffer comprisinghistidine, an organic co-solvent comprising polysorbate, and astabilizing agent comprising a sugar, an amino acid or both, whereinsaid VEGF antagonist fusion protein is capable of inhibiting biologicalactivity of human VEGF as measured by a mouse Baf/3 VEGFR1/EpoR cellline and achieving a percent relative potency of at least 75 relative toa reference VEGF IC₅₀ standard.
 28. The formulation of claim 27, whereinsaid buffer comprises 5-50 mM histidine.
 29. The formulation of claim27, wherein said percent relative potency of at least 75 is achievedafter 1 month of storage at 5° C.
 30. The formulation of claim 27,wherein said percent relative potency of at least 75 is achieved after24 months of storage at 5° C.
 31. A formulation comprising: 10-50 mg/mlof a vascular endothelial growth factor (VEGF) antagonist fusion proteincomprising amino acids 27-457 of SEQ ID NO: 4, a buffer comprisinghistidine, an organic co-solvent comprising polysorbate, and astabilizing agent comprising a sugar, an amino acid or both, whereinsaid VEGF antagonist fusion protein is capable of binding VEGF at apercent relative binding of at least 88 relative to a reference VEGFIC₅₀ standard, after storage at 5° C. for 3 months.
 32. The formulationof claim 31, wherein said buffer comprises 5-50 mM histidine.
 33. Amethod of manufacturing a liquid formulation, said method comprisingcombining a vascular endothelial growth factor (VEGF) antagonist fusionprotein that comprises amino acids 27-457 of SEQ ID NO: 4 with a buffercomprising histidine, an organic co-solvent comprising polysorbate, anda stabilizing agent comprising a sugar, an amino acid or both; whereinthe concentration of said VEGF antagonist fusion protein is 10-50 mg/mlin said liquid formulation; and wherein said VEGF antagonist fusionprotein exhibits less than about 3% degradation after 15 months ofstorage at 5° C.
 34. The method of claim 33, wherein said buffercomprises 5-50 mM histidine.
 35. A method of manufacturing a liquidformulation, said method comprising combining a vascular endothelialgrowth factor (VEGF) antagonist fusion protein that comprises aminoacids 27-457 of SEQ ID NO: 4 with a buffer comprising histidine, anorganic co-solvent comprising polysorbate, and a stabilizing agentcomprising a sugar, an amino acid or both; wherein the concentration ofsaid VEGF antagonist fusion protein is 10-50 mg/ml in said liquidformulation; and wherein said VEGF antagonist fusion protein is capableof inhibiting biological activity of human VEGF as measured by a mouseBaf/3 VEGFR1/EpoR cell line and achieving a percent relative potency ofat least 75 relative to a reference VEGF IC₅₀ standard.
 36. The methodof claim 35, wherein said buffer comprises 5-50 mM histidine.
 37. Themethod of claim 35, wherein said percent relative potency of at least 75is achieved after 1 month of storage at 5° C.
 38. The formulation ofclaim 35, wherein said percent relative potency of at least 75 isachieved after 24 months of storage at 5° C.
 39. A method ofmanufacturing a liquid formulation, said method comprising combining avascular endothelial growth factor (VEGF) antagonist fusion protein thatcomprises amino acids 27-457 of SEQ ID NO: 4 with a buffer comprisinghistidine, an organic co-solvent comprising polysorbate, and astabilizing agent comprising a sugar, an amino acid or both; wherein theconcentration of said VEGF antagonist fusion protein is 10-50 mg/ml insaid liquid formulation; and wherein said VEGF antagonist fusion proteinis capable of binding VEGF at a percent relative binding of at least 88relative to a reference VEGF IC₅₀ standard, after storage at 5° C. for 3months.
 40. The method of claim 39, wherein said buffer comprises 5-50mM histidine.
 41. The formulation of claim 2, wherein said organicco-solvent comprises 0.05-0.15% polysorbate
 20. 42. The formulation ofclaim 2, wherein said organic co-solvent comprises 0.003-0.15%polysorbate.
 43. The formulation of claim 29, wherein said buffercomprises 5-50 mM histidine.
 44. The formulation of claim 29, whereinsaid buffer comprises about 10 mM histidine.
 45. The formulation ofclaim 43, wherein said buffer is at a pH of about 6.0-6.5.
 46. Theformulation of claim 43, wherein said formulation comprises 40 mg/ml ofsaid VEGF antagonist fusion protein.
 47. The formulation of claim 46,wherein said formulation further comprises a tonicity agent.
 48. Theformulation of claim 47, wherein said tonicity agent is sodium chloride.49. The formulation of claim 46, wherein said organic co-solventcomprises 0.1-0.5% polysorbate.
 50. The formulation of claim 46, whereinsaid organic co-solvent comprises 0.05-0.15% polysorbate
 20. 51. Theformulation of claim 46, wherein said organic co-solvent comprises0.003-0.15% polysorbate.
 52. The formulation of claim 46, wherein saidstabilizing agent comprises a sugar.
 53. The formulation of claim 52,wherein said sugar is selected from group consisting of dextrose,ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose, andlactose.
 54. The formulation of claim 52, wherein said sugar is 5-20%sucrose.
 55. The formulation of claim 52, wherein said sugar is 0.5-6.0%sucrose.
 56. The formulation of claim 46, wherein said stabilizing agentcomprises an amino acid.
 57. The formulation of claim 56, wherein saidamino acid is glycine.
 58. The formulation of claim 31, wherein saidbuffer comprises about 10 mM histidine.
 59. The formulation of claim 32,wherein said buffer is at a pH of about 6.0-6.5.
 60. The formulation ofclaim 32, wherein said formulation comprises 40 mg/ml of said VEGFantagonist fusion protein.
 61. The formulation of claim 60, wherein saidformulation further comprises a tonicity agent.
 62. The formulation ofclaim 61, wherein said tonicity agent is sodium chloride.
 63. Theformulation of claim 60, wherein said organic co-solvent comprises0.1-0.5% polysorbate.
 64. The formulation of claim 60, wherein saidorganic co-solvent comprises 0.05-0.15% polysorbate
 20. 65. Theformulation of claim 60, wherein said organic co-solvent comprises0.003-0.15% polysorbate.
 66. The formulation of claim 60, wherein saidstabilizing agent comprises a sugar.
 67. The formulation of claim 66,wherein said sugar is selected from group consisting of dextrose,ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose, andlactose.
 68. The formulation of claim 66, wherein said sugar is 5-20%sucrose.
 69. The formulation of claim 66, wherein said sugar is 0.5-6.0%sucrose.
 70. The formulation of claim 60, wherein said stabilizing agentcomprises an amino acid.
 71. The formulation of claim 70, wherein saidamino acid is glycine.
 72. The method of claim 33, wherein said buffercomprises about 10 mM histidine.
 73. The method of claim 34, whereinsaid buffer is at a pH of about 6.0-6.5.
 74. The method of claim 34,wherein said formulation comprises 40 mg/ml of said VEGF antagonistfusion protein.
 75. The method of claim 74, wherein said formulationfurther comprises a tonicity agent.
 76. The method of claim 75, whereinsaid tonicity agent is sodium chloride.
 77. The method of claim 74,wherein said organic co-solvent comprises 0.1-0.5% polysorbate.
 78. Themethod of claim 74, wherein said organic co-solvent comprises 0.05-0.15%polysorbate
 20. 79. The method of claim 74, wherein said organicco-solvent comprises 0.003-0.15% polysorbate.
 80. The method of claim74, wherein said stabilizing agent comprises a sugar.
 81. The method ofclaim 80, wherein said sugar is selected from group consisting ofdextrose, ribose, fructose, mannitol, inositol, sorbitol, trehalose,sucrose, and lactose.
 82. The method of claim 80, wherein said sugar is5-20% sucrose.
 83. The method of claim 80, wherein said sugar is0.5-6.0% sucrose.
 84. The method of claim 74, wherein said stabilizingagent comprises an amino acid.
 85. The method of claim 84, wherein saidamino acid is glycine.
 86. The method of claim 37, wherein said buffercomprises 5-50 mM histidine.
 87. The method of claim 37, wherein saidbuffer comprises about 10 mM histidine.
 88. The method of claim 86,wherein said buffer is at a pH of about 6.0-6.5.
 89. The method of claim86, wherein said formulation comprises 40 mg/ml of said VEGF antagonistfusion protein.
 90. The method of claim 89, wherein said formulationfurther comprises a tonicity agent.
 91. The method of claim 90, whereinsaid tonicity agent is sodium chloride.
 92. The method of claim 89,wherein said organic co-solvent comprises 0.1-0.5% polysorbate.
 93. Themethod of claim 89, wherein said organic co-solvent comprises 0.05-0.15%polysorbate
 20. 94. The method of claim 89, wherein said organicco-solvent comprises 0.003-0.15% polysorbate.
 95. The method of claim89, wherein said stabilizing agent comprises a sugar.
 96. The method ofclaim 95, wherein said sugar is selected from group consisting ofdextrose, ribose, fructose, mannitol, inositol, sorbitol, trehalose,sucrose, and lactose.
 97. The method of claim 95, wherein said sugar is5-20% sucrose.
 98. The method of claim 95, wherein said sugar is0.5-6.0% sucrose.
 99. The method of claim 89, wherein said stabilizingagent comprises an amino acid.
 100. The method of claim 99, wherein saidamino acid is glycine.
 101. The method of claim 39, wherein said buffercomprises about 10 mM histidine.
 102. The method of claim 40, whereinsaid buffer is at a pH of about 6.0-6.5.
 103. The method of claim 40,wherein said formulation comprises 40 mg/ml of said VEGF antagonistfusion protein.
 104. The method of claim 103, wherein said formulationfurther comprises a tonicity agent.
 105. The method of claim 104,wherein said tonicity agent is sodium chloride.
 106. The method of claim103, wherein said organic co-solvent comprises 0.1-0.5% polysorbate.107. The method of claim 103, wherein said organic co-solvent comprises0.05-0.15% polysorbate
 20. 108. The method of claim 103, wherein saidorganic co-solvent comprises 0.003-0.15% polysorbate.
 109. The method ofclaim 103, wherein said stabilizing agent comprises a sugar.
 110. Themethod of claim 109, wherein said sugar is selected from groupconsisting of dextrose, ribose, fructose, mannitol, inositol, sorbitol,trehalose, sucrose, and lactose.
 111. The method of claim 109, whereinsaid sugar is 5-20% sucrose.
 112. The method of claim 109, wherein saidsugar is 0.5-6.0% sucrose.
 113. The method of claim 103, wherein saidstabilizing agent comprises an amino acid.
 114. The method of claim 113,wherein said amino acid is glycine.